Fluorescence Lifetime Imaging Microscopy (FLIM) Data Analysis with TIMP

Fluorescence Lifetime Imaging Microscopy (FLIM) allows fluorescence lifetime images of biological objects to be collected at 250 nm spatial resolution and at (sub-)nanosecond temporal resolution. Often n_comp kinetic processes underlie the observed fluorescence at all locations, but the intensity of the fluorescence associated with each process varies per-location, i.e., per-pixel imaged. Then the statistical challenge is global analysis of the image: use of the fluorescence decay in time at all locations to estimate the n_comp lifetimes associated with the kinetic processes, as well as the amplitude of each kinetic process at each location. Given that typical FLIM images represent on the order of 10^2 timepoints and 10^3 locations, meeting this challenge is computationally intensive. Here the utility of the TIMP package for R to solve parameter estimation problems arising in FLIM image analysis is demonstrated. Case studies on simulated and real data evidence the applicability of the partitioned variable projection algorithm implemented in TIMP to the problem domain, and showcase options included in the package for the visual validation of models for FLIM data.

On the Molecular and Submolecular Structure of the Semiquinone Cations of Alloxazines and Isoalloxazines as Revealed by Electron-Paramagnetic-Resonance Spectroscopy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66144/1/j.1432-1033.1981.tb05295.x.pd

Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λmax) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages

Time-resolved fluorescence study of the dissociation of FMN from the yellow fluorescence protein from Vibrio fischeri

Time-resolved fluorescence spectroscopy of the flavin mononucleotide (FMN) prosthetic group of the yellow fluorescence protein (YFP) from Vibrio fischeri has provided quantitative, thermodynamic information on the FMN-apoYFP equilibrium in aqueous buffer. In diluted aqueous solution two fluorescent species could be identified by distinct fluorescence lifetimes and rotational correlation times originating from free- and protein-bound FMN. Quantitation of the amounts of free and bound FMN in progressively larger dilutions of YFP in aqueous buffer yielded a dissociation constant of 0.40 fJ.M for the FMN-apoprotein complex at 20°C. The single fluorescence lifetime of YFP-bound FMN is very long (7.6 ns at 20°C), suggesting a binding environment in which maximal emission is provided commensurate with its function as a bioluminescent emitter. The single correlation time of 14.8 ns (20°C) is in agreement with a rigid binding site that rotates together with the whole, hydrated protein. Using a different technique we have obtained the same results as reported by others (G. Sirokman, T. Wilson and J. W. Hastings, Biochemistry 34, 13074-13081, 1995; V. N. Petushkov, B. G. Gibson and J. Lee, Biochem.Biophys. Res. Commun. 211,774-779,1995)

Time-resolved fluorescence study of azurin variants: conformational heterogeneity and tryptophan mobility. Biophys

ABSTRACT Time-resolved fluorescence and time resolved fluorescence anisotropy studies have been performed on wild-type azurin from Pseudomonas aeruginosa and two variants to study the mobility of Trp 48 . The two azurin variants in which the microenvironment of Trp 48 was changed comprised the single mutations Ile 7 Ser and Phe 110 Ser. The experiments were performed on the holo-Cu(I), holo-Cu(II), and apo-forms at various pH values, viscosities, and temperatures; two distinct parts of the emission spectrum were selected for detection. Two prominent subnanosecond lifetimes in the fluorescence decays of the Cu(II) proteins could be observed. The decay of apo-azurin also consists of more than one component. The occurrence of more than one component in the fluorescence decays is explained by conformational heterogeneity

Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins

Abstract Visible fluorescent proteins from Aequorea victoria contain next to the fluorophoric group a single tryptophan residue. Both molecules form a single donor -acceptor pair for resonance energy transfer (RET) within the protein. Time-resolved fluorescence experiments using tryptophan excitation have shown that RET is manifested by a distinct growing in of acceptor fluorescence at a rate characteristic for this process. In addition, time-resolved fluorescence anisotropy measurements under the same excitation -emission conditions showed a correlation time that is similar to the time constant of the same RET process with the additional benefit of gaining information on the relative orientation of the corresponding transition dipoles.